The objective of this proposed research is to establish limbic and hypothalamic sites of interaction between gonadal steroids and neuronal substance P (SP) as a means of understanding how SP may participate in regulation of reproductive function. Evidence is accumulating that SP, like other neuroactive peptides in the hypothalamus, is subject to both organizational and activational influences of gonadal steroids. Sex differences in regional distribution of SP have been demonstrated in the hypothalamus and amygdala although these differences have not been resolved at the cellular level. Fluctuations of SP content and neurosecretory activity of SP immunoreactive neurons during the estrous cycle suggests that hypothalamic cells that express SP may be targets of estrogen activation. We propose to test for steroid regulation of SP by comparing distributions of SP immunoreactive elements in males and females and to test the steroid dependency of the SP system by comparing these distributions in gonadectomized rats with and without steroid replacement. Our preliminary studies employing the combined techniques of autoradiography and immunohistochemistry has revealed a population of SP immunoreactive neurons which concentrate estradiol in the ventromedial and arcuate nuclei and the lateral hypothalamus. This finding provides direct support for the hypothesis that gonadal steroids activate neurons which express this peptide. We plan to confirm and extend this finding to other areas where the distribution of steroid concentration overlaps SP immunoreactivity including the medial nucleus of the amygdala, bed nucleus of the stria terminalis and the preoptic area. The retrograde tracer, fluorogold, will be used to test for the extent of SP-specific connectivity among these structures. Although the hypothalamus contains an abundant population of neurons that express SP and this excitatory peptide exhibits characteristics of a neurotransmitter/modulator, the function of hypothalamic SP is still largely unknown. Cerebroventricular injections of SP prolong the estrous cycle and stimulate release of luteinizing hormone, siggestomg a role in processes that control reporductive funciton. Thus hypothalamic neurons that express SP appear to be prime candidates for study of the link between gonadal steroid feedback and mechanisms which are essential for the regulation of reproduction. Findings generated by the proposed research will contribute significant new information concerning the interaction between gonadal steroids and neuropeptides.